Diabetes is characterized by prolonged hyperglycaemic conditions resulting from pancreatic dysfunctions thereby causing deficiencies in insulin secretion, insulin action, or both and leading to abnormalities in carbohydrate, fat, and protein metabolism. One of the several consequences of elevated plasma glucose levels in diabetics is enhanced protein glycation. Glycation involves non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is stimulated by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins including human serum albumin (HSA). Human serum albumin is an abundant plasma protein present in concentrations ranging from 30-50 g/L and accounts for approximately 60% of the total protein content in serum.
Biomarkers associated with diabetes are mainly based on protein glycation e.g. glycated hemoglobin (HbA1c), fructosamine and glycated albumin. HbA1c is considered as a ‘gold standard’ marker reflecting the glycemic status of a diabetic over a period of 90-120 days. Although many studies have suggested the usefulness of HbA1c, factors such as anaemia, blood loss, splenomegaly, iron deficiency can cause severe fluctuations in levels of HbA1c (Radin M S. J Gen Intern Med 2014; 29:388-94). In view of HbA1c level being influenced by haemodialysis, hemoglobin level, and erythropoietin dose, glycated albumin levels could be a more reliable indicator of glycemic level in diabetics and in patients on hemodialysis who have diabetes and anuria than HbA1c level. Alternatively, glycated albumin has also been proposed as a glycemic biomarker, as it reflects short-term changes in glucose levels, and has been strongly recommended for gestational diabetes (Hashimoto K et al, Diabetes Care 2010; 33:509-11). Juraschek S P, et al (Diabetes Care 2012; 35:2265-70) and Bhonsle H S, et al (J Proteome Res 2012; 11:1391-6) have studied positive correlation between increased levels of glycated albumin and hyperglycaemia.
Additionally, several recent studies have suggested that levels of glycated albumin are associated with pre-diabetic conditions/symptoms, and microalbuminuria. Thus, the quantification of glycated albumin is of appreciable clinical significance.
Human serum albumin readily undergoes glycation contributing mainly to increased levels of advanced glycation end-products in plasma, thus glycated albumin has been suggested as an additional marker for monitoring the glycemic status. Several studies have implicated AGE's in the development of insulin resistance, as well as in pathogenesis of diabetic complications (Manigrasso M B et al, Trends Endocrin Met 2014; 25:15-22). Certain lysine residues of HSA are more prone to undergo glycation modification, these are also referred to as glycation/glucose sensitive sites (Thornalley, P. J., et al (2003) Biochem J 375, 581-592). Since these lysine residues are constantly exposed to higher glucose concentration; the specific sites can undergo sequential AGE modifications followed by initial Amadori rearrangement. Further, CML and CEL constitute around 80% percent of total AGE's. The levels of AGEs increase substantially in diabetic plasma due to hyperglycemic condition. Therefore, analysis of plasma AGEs can possibly provide information about the severity of diabetes.
Prevalent quantification techniques are restricted to estimate Amadori modified lysine (AML) peptides of albumin, however there has been no method devised to quantify levels of predominant AGE modified peptides such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) peptides of albumin which are required to be quantified in diabetics to gauge the extent of damage and complications arisen by the disease.
Methods reported to quantify AGE modified albumin by various approaches include colorimetric assay, ketoamine oxidase assay, enzyme-linked boronate immunoassay, fluorescence spectroscopy, boronic acid affinity chromatography assay and mass spectrometry (MS). Amongst these approaches, MS offers precise characterization of protein glycation including the amino acid involved in the modification. AML modification has been extensively studied by different MS approaches. The fragmentation pattern and diagnostic ions for AML rearrangement product has been well established.
Diagnostic ions serve as the most reliable way of identifying glycated peptide by tandem mass spectrometry. Thus, having a good MS/MS fragment ions is key for precise characterization of glycation. However, the ratio of in vivo AGE modified to unmodified protein is significantly low, which limits better MS/MS. Further, Zhang Q B, et al (J Proteome Res 2011; 10:3076-88) by using a combination of immune-depletion, enrichment and fractionation strategies, have identified a total of 7749 unique glycated peptides corresponding to 1095 native human plasma proteins, 1592 in vitro glycated human plasma proteins and 1664 erythrocyte proteins.
Stable-isotope-dilution tandem mass spectrometry method has also been employed for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins and 13C6-glucose to quantify glycated proteins in the plasma and erythrocytes. In a recent study, the glycation sensitive peptides of HSA that could serve as markers for early diagnosis of type 2 diabetes were quantified by using MS based 18O-labeling technique (Zhang M et al, Diabetes 2013; 62:3936-42). However, most of the previous studies have focused on AML modification, than other AGE modification. Therefore, there arises a need for the development of MS/MS fragment ion library for quantification of AML, CML and CEL modifications of albumin.
Hoffmann R et al in a research study published in J Proteome Res. 2015 Feb. 6; 14(2): 768-77 reported characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification. The process employed allowed sensitive and selective precursor ion scans to detect modified peptides in complex sample mixtures. Even though, this study teaches the CML modified sites in different proteins, including modified lysine residues 88 and 396 of human serum albumin, there is no attempt to identify specific modified peptides in any particular stage of diabetes. Moreover, glycation is chronic process; a given protein can undergo dynamic heterogeneous transformations as they have varying biological life span influencing the function of a protein. Thus, to assess the degree of glycation at a given pathophysiological condition, precise identification of glycation becomes critical.
Since there is a pending need in the art to understand AGE site specific modification in albumin and keeping in mind the clinical significance of AGE-HSA quantification, the present inventors have devised a method for identifying and quantifying diagnostic peptide markers specific to AGE modified human serum albumin, mainly carboxymethyl and carboxyethyl lysine residues.